Cloned DNA polymerases from Thermotoga neapolitana

ABSTRACT

The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga neapolitana (Tne). The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to the cloning and expression of the Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The Tne DNA polymerase of the invention may be used in well-known DNA sequencing and amplification reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/316,423, filed Sep. 30, 1994, titled "Cloned DNA Polymerases fromThermotoga neapolitana," the contents of which are incorporated hereinin their entirety by reference, and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a substantially pure thermostable DNApolymerase. Specifically, the DNA polymerase of the present invention isa Thermotoga neapolitana DNA polymerase having a molecular weight ofabout 100 kilodaltons. The present invention also relates to cloning andexpression of the Thermotoga neapolitana DNA polymerase in E. coli, toDNA molecules containing the cloned gene, and to hosts which expresssaid genes. The DNA polymerase of the present invention may be used inDNA sequencing and amplification reactions.

2. Background Information

DNA polymerases synthesize the formation of DNA molecules which arecomplementary to a DNA template. Upon hybridization of a primer to thesingle-stranded DNA template, polymerases synthesize DNA in the 5' to 3'direction, successively adding nucleotides to the 3'-hydroxyl group ofthe growing strand. Thus, in the presence of deoxyribonucleosidetriphosphates (dNTPs) and a primer, a new DNA molecule, complementary tothe single stranded DNA template, can be synthesized.

A number of DNA polymerases have been isolated from mesophilicmicroorganisms such as E. coli. A number of these mesophilic DNApolymerases have also been cloned. Lin et al. cloned and expressed T4DNA polymerase in E. coli (Proc. Natl. Acad. Sci. USA 84:7000-7004(1987)). Tabor et al. (U.S. Pat. No. 4,795,699) describes a cloned T7DNA polymerase, while Minkley et al. (J. Biol. Chem. 259:10386-10392(1984)) and Chatterjee (U.S. Pat. No. 5,047,342) described E. coli DNApolymerase I and cloning of T5 DNA polymerase, respectively.

Although DNA polymerases from thermophiles are known, relatively littleinvestigation has been done to isolate and even clone these enzymes.Chien et al., J. Bacteriol. 127:155-1557 (1976) describe a purificationscheme for obtaining a polymerase from Thermus aquaticus. The resultingprotein had a molecular weight of about 63,000 daltons by gel filtrationanalysis and 68,000 daltons by sucrose gradient centrifugation. Kaledinet al., Biokhymiya 45:644-51 (1980) disclosed a purification procedurefor isolating DNA polymerase from T. aquaticus YET1 strain. The purifiedenzyme was reported to be a 62,000 dalton monomeric protein. Gelfand etal. (U.S. Pat. No. 4,889,818) cloned a gene encoding a thermostable DNApolymerase from Thermus aquaticus. The molecular weight of this proteinwas found to be about 86,000 to 90,000 daltons.

Simpson et al. purified and partially characterized a thermostable DNApolymerase from a Thermotoga species (Biochem. Cell. Biol. 86:1292-1296(1990)). The purified DNA polymerase isolated by Simpson et al.exhibited a molecular weight of 85,000 daltons as determined bySDS-polyacrylamide gel electrophoresis and size-exclusionchromatography. The enzyme exhibited half-lives of 3 minutes at 95° C.and 60 minutes at 50° C. in the absence of substrate and its pH optimumwas in the range of pH 7.5 to 8.0. Triton X-100 appeared to enhance thethermostability of this enzyme. The strain used to obtain thethermostable DNA polymerase described by Simpson et al. was Thermotogaspecies strain FjSS3-B.1 (Hussar et al., FEMS Microbiology Letters37:121-127 (1986)). Other DNA polymerases have been isolated fromthermophilic bacteria including Bacillus steraothermophilus (Stenesh etal., Biochim. iochys. Acta 272:156-166 (1972); and Kaboev et al., J.Bacteriol. 145:21-26 (1981)) and several archaebacterial species (Rossiet al., System. Appl. Microbiol. 7:337-341 (1986); Klimczak et al.,Biochemistry 25:4850-4855 (1986); and Elie et al., Eur. J. Biochem.178:619-626 (1989)). The most extensively purified archaebacterial DNApolymerase had a reported half-life of 15 minutes at 87° C. (Elie et al.(1989), supra). Innis et al., In PCR Protocol: A Guide To Methods andAmplification, Academic Press, Inc., San Diego (1990) noted that thereare several extreme thermophilic eubacteria and archaebacteria that arecapable of growth at very high temperatures (Bergquist et al., Biotech.Genet. Eng. Rev. 5:199-244 (1987); and Kelly et al., Biotechnol Prog.4:47-62 (1988)) and suggested that these organisms may contain verythermostable DNA polymerases.

SUMMARY OF THE INVENTION

The present invention is directed to a thermostable DNA polymerasehaving a molecular weight of about 100 kilodaltons. More specifically,the DNA polymerase of the invention is isolated from Thermotoganeapolitana (Tne). The Thermotoga species preferred for isolating theDNA polymerase of the present invention was isolated from an Africancontinental solfataric spring (Windberger et al., Arch. Microbiol. 151.506-512, (1989)).

The Tne DNA polymerase of the present invention is extremelythermostable, showing more than 50% of activity after being heated for60 minutes at 90° C. with or without detergent. Thus, the DNA polymeraseof the present invention is more thermostable than Taq DNA polymerase.

The present invention is also directed to cloning a gene encoding aThermotoga neapolitana DNA polymerase enzyme. DNA molecules containingthe Tne DNA polymerase gene, according to the present invention, can betransformed and expressed in a host cell to produce a Tne DNA polymerasehaving a molecular weight of 100 kilodaltons. Any number of hosts may beused to express the Thermotoga DNA polymerase gene of the presentinvention; including prokaryotic and eukaryotic cells. Preferably,prokaryotic cells are used to express the DNA polymerase of theinvention. The preferred prokaryotic hosts according to the presentinvention is E. coli.

The Tne DNA polymerase of the invention may be used in well known DNAsequencing (dideoxy DNA sequencing, cycle DNA sequencing of plasmidDNAs, etc.) and DNA amplification reactions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates the heat stability of Tne DNA polymerase at 90° C.over time. Crude extract from Thermotoga neapolitana cells was used inthe assay.

FIG. 2 shows the DNA polymerase activity in crude extracts from an E.coli host containing the cloned Tne DNA polymerase gene.

FIG. 3 compares the ability of various DNA polymerases to incorporateradioactive dATP and αS!dATP. Tne DNA polymerase is more effective atincorporating αS!dATP than was Taq DNA polymerase.

FIG. 4 shows the restriction map of the approximate DNA fragment whichcontains the Tne DNA polymerase gene in pSport 1 and pUC19.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

In the description that follows, a number of terms used in recombinantDNA technology are extensively utilized. In order to provide a clearerand consistent understanding of the specification and claims, includingthe scope to be given such terms, the following definitions areprovided.

Cloning vector. A plasmid, cosmid or phage DNA or other DNA moleculewhich is able to replicate autonomously in a host cell, and which ischaracterized by one or a small number of endonuclease recognition sitesat which such DNA sequences may be cut in a determinable fashion withoutloss of an essential biological function of the vector, and into whichDNA may be spliced in order to bring about its replication and cloning.The cloning vector may further contain a marker suitable for use in theidentification of cells transformed with the cloning vector. Markers,for example, are tetracycline resistance or ampicillin resistance.

Expression vector. A vector similar to a cloning vector but which iscapable of enhancing the expression of a gene which has been cloned intoit, after transformation into a host. The cloned gene is usually placedunder the control of (i.e., operably linked to) certain controlsequences such as promoter sequences.

Recombinant host. Any prokaryotic or eukaryotic or microorganism whichcontains the desired cloned genes on an expression vector, cloningvector or any DNA molecule. The term "recombinant host" is also meant toinclude those host cells which have been genetically engineered tocontain the desired gene on the host chromosome or genome.

Host. Any prokaryotic or eukaryotic microorganism that is the recipientof a replicable expression vector, cloning vector or any DNA molecule.The DNA molecule may contain, but is not limited to, a structural gene,a promoter and/or an origin of replication.

Promoter. A DNA sequence generally described as the 5' region of a gene,located proximal to the start codon. At the promoter region,transcription of an adjacent gene(s) is initiated.

Gene. A DNA sequence that contains information necessary for expressionof a polypeptide or protein. It includes the promoter and the structuralgene as well as other sequences involved in expression of the protein.

Structural gene. A DNA sequence that is transcribed into messenger RNAthat is then translated into a sequence of amino acids characteristic ofa specific polypeptide.

Operably linked. As used herein means that the promoter controls theinitiation of the expression of the polypeptide encoded by thestructural gene.

Expression. Expression is the process by which a gene produces apolypeptide. It involves transcription of the gene into messenger RNA(mRNA) and the translation of such mRNA into polypeptide(s).

Substantially Pure. As used herein "substantially pure" means that thedesired purified protein is essentially free from contaminating cellularcontaminants which are associated with the desired protein in nature.Contaminating cellular components may include, but are not limited to,phosphatases, exonucleases, endonucleases or undesirable DNA polymeraseenzymes.

Primer. As used herein "primer" refers to a single-strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a DNA molecule.

Template. The term "template" as used herein refers to a double-strandedor single-stranded DNA molecule which is to be amplified, synthesized orsequenced. In the case of a double-stranded DNA molecule, denaturationof its strands to form a first and a second strand is performed beforethese molecules may be amplified, synthesized or sequenced. A primer,complementary to a portion of a DNA template is hybridized underappropriate conditions and the DNA polymerase of the invention may thensynthesize a DNA molecule complementary to said template or a portionthereof. The newly synthesized DNA molecule, according to the invention,may be equal or shorter in length than the original DNA template.Mismatch incorporation during the synthesis or extension of the newlysynthesized DNA molecule may result in one or a number of mismatchedbase pairs. Thus, the synthesized DNA molecule need not be exactlycomplementary to the DNA template.

Incorporating. The term "incorporating" as used herein means becoming apart of a DNA molecule or primer.

Amplification. As used herein "amplification" refers to any in vitromethod for increasing the number of copies of a nucleotide sequence withthe use of a DNA polymerase. Nucleic acid amplification results in theincorporation of nucleotides into a DNA molecule or primer therebyforming a new DNA molecule complementary to a DNA template. The formedDNA molecule and its template can be used as templates to synthesizeadditional DNA molecules. As used herein, one amplification reaction mayconsist of many rounds of DNA replication. DNA amplification reactionsinclude, for example, polymerase chain reactions (PCR). One PCR reactionmay consist of 30 to 100 "cycles" of denaturation and synthesis of a DNAmolecule.

Oligonucleotide. "Oligonucleotide" refers to a synthetic or naturalmolecule comprising a covalently linked sequence of nucleotides whichare joined by a phosphodiester bond between the 3' position of thepentose of one nucleotide and the 5' position of the pentose of theadjacent nucleotide.

Nucleotide. As used herein "nucleotide" refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes deoxyribonucleosidetriphosphates such as dATP, dCTP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives include, for example, dm, αS!dATP and7-deaza-dGTP. The term nucleotide as used herein also refers todideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.Illustrated examples of dideoxyribonucleoside triphosphates include, butare not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According tothe present invention, a "nucleotide" may be unlabeled or detectablylabeled by well known techniques. Detectable labels include, forexample, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels.

Thermostable. As used herein "thermostable" refers to a DNA polymerasewhich is resistant to inactivation by heat. DNA polymerases synthesizethe formation of a DNA molecule complementary to a single-stranded DNAtemplate by extending a primer in the 5' to 3' direction. This activityfor mesophilic DNA polymerases may be inactivated by heat treatment. Forexample, T5 DNA polymerase activity is totally inactivated by exposingthe enzyme to a temperature of 90° C. for 30 seconds. As used herein, athermostable DNA polymerase activity is more resistant to heatinactivation than a mesophilic DNA polymerase. However, a thermostableDNA polymerase does not mean to refer to an enzyme which is totallyresistant to heat inactivation and thus heat treatment may reduce theDNA polymerase activity to some extent. A thermostable DNA polymerasetypically will also have a higher optimum temperature than mesophilicDNA polymerases.

Hybridization. The terms "hybridization" and "hybridizing" refers to thepairing of two complementary single-stranded nucleic acid molecules (RNAand/or DNA) to give a double-stranded molecule. As used herein, twonucleic acid molecules may be hybridized, although the base pairing isnot completely complementary. Accordingly, mismatched bases do notprevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used.

A. Cloning and Expression of Thermotoga neapolitana DNA Polymerase

The Thermotoga DNA polymerase of the invention can be isolated from anystrain of Thermotoga which produces a DNA polymerase having themolecular weight of about 100 kilodaltons. The preferred strain toisolate the gene encoding Thermotoga DNA polymerase of the presentinvention is Thermotoga neapolitana. The most preferred Thermotoganeapolitana for isolating the DNA polymerase of the invention wasisolated from an African continental solfataric spring (Windberger etal., Arch. Microbiol. 151:506-512 (1989) and may be obtained fromDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM; GermanCollection of Microorganisms and Cell Culture) Mascheroder Weg 1b D-3300Braunschweig, Federal Republic of Germany, as Deposit No. 5068.

To clone a gene encoding the Thermotoga neapolitana DNA polymerase ofthe invention, isolated DNA which contains the polymerase gene, obtainedfrom Thermotoga neapolitana cells, is used to construct a recombinantDNA library in a vector. Any vector, well known in the art, can be usedto clone the Thermotoga neapolitana DNA polymerase of the presentinvention. However, the vector used must be compatible with the host inwhich the recombinant DNA library will be transformed.

Prokaryotic vectors for constructing the plasmid library includeplasmids such as those capable of replication in E. coli such as, forexample, pBR322, ColE1, pSC101, pUC-vectors (pUC18, pUC19, etc.: In:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982); and Sambrook et al., In:Molecular Cloning A Laboratory Manual (2d ed.) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). Bacillus plasmidsinclude pC194, pC221, pC217, etc. Such plasmids are disclosed byGlyczan, T. In: The Molecular Biology Bacilli, Academic Press, York(1982), 307-329. Suitable Streptomyces plasmids include pIJ101 (Kendallet al., J. Bacteriol 169:4177-4183 (1987)). Pseudomonas plasmids arereviewed by John et al., (Rad. Insec. Dis0. 8:693-704 (1986)), andIgaki, (Jpn. J. Bacteriol. 33:729-742 (1978)). Broad-host range plasmidsor cosmids, such as pCP13 (Darzins and Chakrabarbary, J. Bacteriol.159:9-18, 1984) can also be used for the present invention. Thepreferred vectors for cloning the genes of the present invention areprokaryotic vectors. Preferably, pCP13 and pUC vectors are used to clonethe genes of the present invention.

The preferred host for cloning the DNA polymerase gene of the inventionis a prokaryotic host. The most preferred prokaryotic host is E. coli.However, the DNA polymerase gene of the present invention may be clonedin other prokaryotic hosts including, but not limited to, Escherichia,Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and Proteus.Bacterial hosts of particular interest include E. coli DH10B, which maybe obtained from Life Technologies, Inc. (Gaithersburg, Md.).

Eukaryotic hosts for cloning and expression of the DNA polymerase of thepresent invention include yeast, fungi, and mammalian cells. Expressionof the desired DNA polymerase in such eukaryotic cells may require theuse of eukaryotic regulatory regions which include eukaryotic promoters.Cloning and expressing the DNA polymerase gene of the invention ineukaryotic cells may be accomplished by well known techniques using wellknown eukaryotic vector systems.

Once a DNA library has been constructed in a particular vector, anappropriate host is transformed by well known techniques. Transformedcolonies are plated at a density of approximately 200-300 colonies perpetri dish. Colonies are then screened for the expression of a heatstable DNA polymerase by transferring transformed E. coli colonies tonitrocellulose membranes. After the transferred cells are grown onnitrocellulose (approximately 12 hours), the cells are lysed by standardtechniques, and the membranes are then treated at 95° C. for 5 minutesto inactivate the endogenous E. coli enzyme. Other temperatures may beused to inactivate the host polymerases depending on the host used andthe temperature stability of the DNA polymerase to be cloned. Stable DNApolymerase activity is then detected by assaying for the presence of DNApolymerase activity using well known techniques. The gene encoding a DNApolymerase of the present invention can be cloned using the proceduredescribed by Sagner et al., Gene 97:119-123 (1991), which reference isherein incorporated by reference in its entirety.

The recombinant host containing the gene encoding DNA polymerase, E.coli DH10B (pUC-Tne), was deposited on Sep. 30, 1994, with the PatentCulture Collection, Northern Regional Research Center, USDA, 1815 NorthUniversity Street, Peoria, Ill. 61604 U.S.A. as Deposit No. NRRLB-21338.

If the Tne DNA polymerase has 3'-5' exo activity, this activity may bereduced or eliminated by mutating the Tne DNA polymerase gene. Suchmutations include point mutations, frame shift mutations, deletions andinsertions. Preferably, the region of the gene encoding the 3'-5' exoactivity is deleted using techniques well known in the art (Sambrook etal., (1989) in: Molecular Cloning, A Laboratory Manual (2nd Ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

B. Enhancing Expression of Thermotoga neapolitana DNA Polymerase

To optimize expression of the Thermotoga DNA polymerase of the presentinvention, inducible or constitutive promoters are well known and may beused to express high levels of a polymerase structural gene in arecombinant host. Similarly, high copy number vectors, well known in theart, may be used to achieve high levels of expression. Vectors having aninducible high copy number may also be useful to enhance expression ofThermotoga DNA polymerase in a recombinant host.

To express the desired structural gene in a prokaryotic cell (such as,E. coli, B. subtilis, Pseudomonas, etc.), it is necessary to operablylink the desired structural gene to a functional prokaryotic promoter.However, the natural Thermotoga neapolitana promoter may function inprokaryotic hosts allowing expression of the polymerase gene. Thus, thenatural Thermotoga promoter or other promoters may be used to expressthe DNA polymerase gene. Such other promoters may be used to enhanceexpression and may either be constitutive or regulatable (i.e.,inducible or derepressible) promoters. Examples of constitutivepromoters include the int promoter of bacteriophage λ, and the blapromoter of the β-lactamase gene of pBR322. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P_(L) and PR_(R)), trp, recA, lacZ, lacI, gal, trc, andtac promoters of E. coli. The B. subtilis promoters include α-amylase(Ulmanen et al., J. Bacteriol 162:176-182 (1985)) and Bacillusbacteriophage promoters (Gryczan, T., In: The Molecular Biology ofBacilli, Academic Press, New York (1982)). Streptomyces promoters aredescribed by Ward et al., Mol. Gen. Genet. 203:468478 (1986)).Prokaryotic promoters are also reviewed by Glick, J. Ind. Microbiol.1:277-282 (1987); Cenatiempto, Y., Biochimie 68:505-516 (1986); andGottesman, Ann. Rev. Genet. 18:415-442 (1984). Expression in aprokaryotic cell also requires the presence of a ribosomal binding siteupstream of the gene-encoding sequence. Such ribosomal binding sites aredisclosed, for example, by Gold et al., Ann. Rev. Microbiol. 35:365404(1981).

To enhance the expression of Tne DNA polymerase in a eukaryotic cell,well known eukaryotic promoters and hosts may be used. Preferably,however, enhanced expression of Tne DNA polymerase is accomplished in aprokaryotic host. The preferred prokaryotic host for overexpressing thisenzyme is E. coli.

C. Isolation and Purification of Thermotoga neapolitana DNA Polymerase

The enzyme(s) of the present invention (Thermotoga neapolitana DNApolymerase, Tne) is preferably produced by fermentation of therecombinant host containing and expressing the cloned DNA polymerasegene. However, the Tne DNA polymerase of the present invention may beisolated from any Thermotoga strain which produces the polymerase of thepresent invention. Fragments of the Tne polymerase are also included inthe present invention. Such fragments include proteolytic fragments andfragments having polymerase activity.

Any nutrient that can be assimilated by Thermotoga neapolitana or a hostcontaining the cloned Tne DNA polymerase gene may be added to theculture medium. Optimal culture conditions should be selected case bycase according to the strain used and the composition of the culturemedium. Antibiotics may also be added to the growth media to insuremaintenance of vector DNA containing the desired gene to be expressed.Culture conditions for Thermotoga neapolitana have, for example, beendescribed by Huber et Antibiotics may also be added to the growth mediato insure maintenance of vector DNA containing the desired gene to beexpressed. Culture conditions for Thermotoga neapolitana have, forexample, been described by Huber et al., Arch. Microbiol. 144:324-333(1986). Media formulations are also described in DSM or ATCC Catalogsand Sambrook et al., In: Molecular Cloning, A Laboratory Manual (2nded.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989).

Thermotoga neapolitana and recombinant host cells producing the DNApolymerase of this invention can be separated from liquid culture, forexample, by centrifugation. In general, the collected microbial cellsare dispersed in a suitable buffer, and then broken down by ultrasonictreatment or by other well known procedures to allow extraction of theenzymes by the buffer solution. After removal of cell debris byultracentrifugation or centrifugation, the DNA polymerase can bepurified by standard protein purification techniques such as extraction,precipitation, chromatography, affinity chromatography, electrophoresisor the like. Assays to detect the presence of the DNA polymerase duringpurification are well known in the art and can be used duringconventional biochemical purification methods to determine the presenceof these enzymes.

D. Uses of Thermotoga neapolitana DNA polymerase

The Thermotoga neapolitana DNA polymerase (Tne) of the present inventionmay be used in well known DNA sequencing, DNA labeling, and DNAamplification reactions. As is well known, sequencing reactions (dideoxyDNA sequencing and cycle DNA sequencing of plasmid DNA) require the useof DNA polymerases. Dideoxy-mediated sequencing involves the use of achain-termination technique which uses a specific polymer for extensionby DNA polymerase, a base-specific chain terminator and the use ofpolyacrylamide gels to separate the newly synthesized chain-terminatedDNA molecules by size so that at least a part of the nucleotide sequenceof the original DNA molecule can be determined. Specifically, a DNAmolecule is sequenced by using four separate DNA sequence reactions,each of which contains different base-specific terminators. For example,the first reaction will contain a G-specific terminator, the secondreaction will contain a T-specific terminator, the third reaction willcontain an A-specific terminator, and a fourth reaction may contain aC-specific terminator. Preferred terminator nucleotides includedideoxyribonucleoside triphosphates (ddNTPs) such as ddATP, ddTTP,ddGTP, and ddCTP. Analogs of dideoxyribonucleoside triphosphates mayalso be used and are well known in the art.

When sequencing a DNA molecule, ddNTPs lack a hydroxyl residue at the 3'position of the deoxyribose base and thus, although they can beincorporated by DNA polymerases into the growing DNA chain, the absenceof the 3'-hydroxy residue prevents formation of a phosphodiester bondresulting in termination of extension of the DNA molecule. Thus, when asmall amount of one ddNTP is included in a sequencing reaction mixture,there is competition between extension of the chain and base-specifictermination resulting in a population of synthesized DNA molecules whichare shorter in length than the DNA template to be sequenced. By usingfour different ddNTPs in four separate enzymatic reactions, populationsof the synthesized DNA molecules can be separated by size so that atleast a part of the nucleotide sequence of the original DNA molecule canbe determined. DNA sequencing by dideoxy-nucleotides is well known andis described by Sambrook et al., In: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989). As will be readily recognized, the Tne DNA polymerase of thepresent invention may be used in such sequencing reactions.

As is well known, detectably labeled nucleotides are typically includedin sequencing reactions. Any number of labeled nucleotides can be usedin sequencing (or labeling) reactions, including, but not limited to,radioactive isotopes, fluorescent labels, chemiluminescent labels,bioluminescent labels, and enzyme labels. It has been discovered thatthe Tne DNA polymerase of the present invention may be useful forincorporating αS nucleotides ( αS!dATP, αS!dTTP, αS!dCTP and αS!dGTP)during sequencing (or labeling) reactions. For example, α³⁵ S!dATP, acommonly used detectably labeled nucleotide in sequencing reactions, isincorporated three times more efficiently with the Tne DNA polymerase ofthe present invention, than with Taq DNA polymerase. Thus, the enzyme ofthe present invention is particularly suited for sequencing or labelingDNA molecules with α³⁵ S!dNTPs.

Polymerase chain reaction (PCR), a well known DNA amplificationtechnique, is a process by which DNA polymerase and deoxyribonucleosidetriphosphates are used to amplify a target DNA template. In such PCRreactions, two primers, one complementary to the 3' termini (or near the3'-termini) of the first strand of the DNA molecule to be amplified, anda second primer complementary to the 3' termini (or near the 3'-termini)of the second strand of the DNA molecule to be amplified, are hybridizedto their respective DNA molecules. After hybridization, DNA polymerase,in the presence of deoxyribonucleoside triphosphates, allows thesynthesis of a third DNA molecule complementary to the first strand anda fourth DNA molecule complementary to the second strand of the DNAmolecule to be amplified. This synthesis results in two double strandedDNA molecules. Such double stranded DNA molecules may then be used asDNA templates for synthesis of additional DNA molecules by providing aDNA polymerase, primers, and deoxyribonucleoside triphosphates. As iswell known, the additional synthesis is carried out by "cycling" theoriginal reaction (with excess primers and deoxyribonucleosidetriphosphates) allowing multiple denaturing and synthesis steps.Typically, denaturing of double stranded DNA molecules to form singlestranded DNA templates is accomplished by high temperatures. TheThermotoga DNA polymerase of the present invention is a heat stable DNApolymerase, and thus will survive such thermal cycling during DNAamplification reactions. Thus, the Tne DNA polymerase of the inventionis ideally suited for PCR reactions, particularly where hightemperatures are used to denature the DNA molecules duringamplification.

E. Kits

The Thermotoga neapolitana (Tne) DNA polymerase of the invention issuited for the preparation of a kit. Kits comprising Tne DNA polymerasemay be used for detectably labeling DNA molecules, DNA sequencing, oramplifying DNA molecules by well known techniques, depending on thecontent of the kit. Such kits may comprise a carrying means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, test tubes and the like. Each of such containermeans comprises components or a mixture of components needed to performDNA sequencing, DNA labeling, or DNA amplification.

A kit for sequencing DNA may comprise a number of container means. Afirst container means may, for example, comprise a substantiallypurified sample of Tne DNA polymerase having the molecular weight ofabout 100 kilodaltons. A second container means may comprise one or anumber of types of nucleotides needed to synthesize a DNA moleculecomplementary to DNA template. A third container means may comprise oneor a number different types of dideoxynucleoside triphosphates. Inaddition to the above container means, additional container means may beincluded in the kit which comprise one or a number of DNA primers.

A kit used for amplifying DNA will comprise, for example, a firstcontainer means comprising a substantially pure Tne DNA polymerase andone or a number of additional container means which comprise a singletype of nucleotide or mixtures of nucleotides. Various primers may ormay not be included in a kit for amplifying DNA.

When desired, the kit of the present invention may also includecontainer means which comprise detectably labeled nucleotides which maybe used during the synthesis or sequencing of a DNA molecule. One of anumber of labels may be used to detect such nucleotides. Illustrativelabels include, but are not limited to, radioactive isotopes,fluorescent labels, chemiluminescent labels, bioluminescent labels andenzyme labels.

Having now generally described the invention, the same will be morereadily understood through reference to the following Examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Bacterial Strains And Growth Conditions

Thermotoga neapolitana DSM No. 5068 was grown under anaerobic conditionsas described in the DSM catalog (addition of resazurin, Na₂ S, andsulfur granules while sparging the media with nitrogen) at 85° C. in anoil bath from 12 to 24 hours. The cells were harvested by filtering thebroth through Whatman #1 filter paper. The supernatant was collected inan ice bath and then centrifuged in a refrigerated centrifuge at 8,000rpms for twenty minutes. The cell paste was stored at -70° C. prior tototal genomic DNA isolation.

E. coli strains were grown in 2×LB broth base (Lennox L broth base:GIBCO/BRL) medium. Transformed cells were incubated in SOC (2% tryptone,0.5% yeast extract, yeast 10 mM NaCl, 2.5M KCl, 20 mM glucose, 10 mMMgCl₂, and 10 mM MgSO₄ per liter) before plating. When appropriateantibiotic supplements were 20 mg/l tetracycline and 100 mg/lampicillin. E. coli strain DH10B (Lorow et al., Focus 12:19-20 (1990))was used as host strain. Competent DH10B may be obtained from LifeTechnologies, Inc. (LTI) (Gaithersburg, Md.).

EXAMPLE 2 DNA Isolation

Thermotoga neapolitana chromosomal DNA was isolated from 1.1 g of cellsby suspending the cells in 2.5 ml TNE (50 mM Tris-HCl, pH 8.0, 50 mMNaCl, 10 mM EDTA) and treated with 1% SDS for 10 minutes at 37° C. DNAwas extracted with phenol by gently rocking the lysed cells overnight at4° C. The next day, the lysed cells were extracted withchloroform:isoamyl alcohol. The resulting chromosomal DNA was furtherpurified by centrifugation in a CsCl density gradient. Chromosomal DNAisolated from the density gradient was extracted three times withisopropanol and dialyzed overnight against a buffer containing 10 mMTris-HCl (pH 8.0) and 1 mM EDTA.

EXAMPLE 3 Construction of Genomic Libraries

The chromosomal DNA isolated in Example 2 was used to construct agenomic library in the plasmid pCP13. Briefly, 10 tubes each containing10 μkg of Thermotoga neapolitana chromosomal DNA was digested with 0.01to 10 units of Sau3Al for 1 hour at 37° C. A portion of the digested DNAwas tested in an agarose (1.2%) gel to determine the extent ofdigestion. Samples with less than 50% digestion were pooled, ethanolprecipitated and dissolved in TE. 6.5 μg of partially digestedchromosomal DNA was ligated into 1.5 μg of pCP13 cosmid which had beendigested with BamHI restriction endonuclease and dephosphorylated withcalf intestinal alkaline phosphatase. Ligation of the partially digestedThermotoga DNA and BamHI cleaved pCP13 was carried out with T4 DNAligase at 22° C. for 16 hours. After ligation, about 1 μg of ligated DNAwas packaged using λ-packaging extract (obtained from Life Technologies,Inc., Gaithersburg, Md.). DH10B cells (Life Tech. Inc.) were theninfected with 100 μl of the packaged material. The infected cells wereplated on tetracycline containing plates. Serial dilutions were made sothat approximately 200 to 300 tetracycline resistant colonies wereobtained per plate.

EXAMPLE 4 Screening for Clones Expressing Thermotoga neapolitana DNAPolymerase

Identification of the Thermotoga neapolitana DNA polymerase gene of theinvention was cloned using the method of Sanger et al., Gene 97:119-123(1991) which reference is herein incorporated in its entirety. Briefly,the E. coli tetracycline resistant colonies from Example 3 weretransferred to nitrocellulose membranes and allowed to grow for 12hours. The cells were then lysed with the fumes of chloroform:toluene(1:1) for 20 minutes and dried for 10 minutes at room temperature. Themembranes were then treated at 95° C. for 5 minutes to inactivate theendogenous E. coli enzymes. Surviving DNA polymerase activity wasdetected by submerging the membranes in 15 ml of polymerase reaction mix(50 mM Tris-HCl (pH 8.8), 1 mM MgCl₂, 3 mM β-mercaptoethanol, 10 μMdCTP, dGTP, dTTP, and 15 μCi of 3,000 Ci/mmol α³² P!dATP) for 30 minutesat 65° C.

Using autoradiography, three colonies were identified that expressed aThermotoga neapolitana DNA polymerase. The cells were grown in liquidculture and the protein extract was made by sonication. The presence ofthe cloned thermostable polymerase was confirmed by treatment at 90° C.followed by measurement of DNA polymerase activity by incorporation ofradioactive deoxyribonucleoside triphosphates into acid insoluble DNA.One of the clones, expressing Tne DNA polymerase, contained a plasmiddesignated pCP13-32 was used for further study.

EXAMPLE 5 Subcloning of Tne DNA polymerase

Since the pCP13-32 clone expressing the Tne polymerase gene containsabout 25 kb of T. neapolitana DNA, we attempted to subclone a smallerfragment of the Tne polymerase gene. The molecular weight of the Tnepolymerase purified from E. coli/pCP13-32 was about 100 Kd. Therefore, a2.5-3.0 kb DNA fragment will be sufficient to code for full-lengthpolymerase. A second round of Sau3A partial digestion similar to Example3 was done using pCP13-32 DNA. In this case, a 3.5 kb region was cut outfrom the agarose gel, purified by Gene Clean (BIO 101, LaJolla, Calif.)and ligated into plasmid pSport 1 (Life Technologies, Inc.) which hadbeen linearized with BamHI and dephosphoylated with calf intestinalphosphatase. After ligation, DH10B was transformed and colonies weretested for DNA polymerase activity as described in Example 4. Severalclones were identified that expressed Tne DNA polymerase. One of theclones (pSport-Tne) containing about 3 kb insert was furthercharacterized. A restriction map of the DNA fragment is shown in FIG. 4.Further, a 2.7 Kb Hind III-SstI fragment was subcloned into pUC19 togenerate pUC19-Tne. E. coli/pUC19-Tne also produced Tne DNA polymerase.

The Tne polymerase clone was sequenced by methods known in the art. Thenucleotide sequence obtained of the 5' end prior to the start ATG isshown in SEQ ID NO:1. The nucleotide sequence obtained which encodes theTne polymerase is shown in SEQ ID NO:2. When SEQ ID NO:2 is translatedit does not produce the entire amino acid sequence of the Tne polymerasedue to frame shift errors in the nucleotide sequence set forth in SEQ IDNO:2. However, an amino acid sequence of the Tne polymerase was obtainedby translating all three reading frames of SEQ ID NO:2, comparing thesesequences with known polymerase amino acid sequences, and splicing theTne polymerase sequence together to form the amino acid sequence setforth in SEQ ID NO:3.

EXAMPLE 6 Purification of Thermotoga neapolitana DNA Polymerase from E.coli

Twelve grams of E. coli cells expressing cloned Tne DNA polymerase(DH10B/pSport-Tne) were lysed by sonication (four thirty-second burstswith a medium tip at the setting of nine with a Heat Systems UltrasonicsInc., model 375 sonicator) in 20 ml of ice cold extraction buffer (50 mMTris HCl, pH 7.4, 8% glycerol, 5 mM mercaptoethanol, 10 mM NaCl, 1 mMEDTA, 0.5 mM PMSF). The sonicated extract was heated at 80° C. for 15min. and then cooled in ice for 5 min. 50 mM KCl and PEI (0.4%) wasadded to remove nucleic acids. The extract was centrifuged forclarification. Ammonium sulfate was added at 60%, the pellet wascollected by centrifugation and resuspended in 10 ml of column buffer(25 mM Tris-HCl, pH 7.4, 8% glycerol, 0.5% EDTA, 5 mM 2-mercaptoethanol,10 mM KCl). A Blue-Sepharose (Pharmacia) column, or preferably a Tosoheparin (Tosohaas) column, was washed with 7 column volumes of columnbuffer and eluted with a 15 column volume gradient of buffer A from 10mM to 2M KCl. Fractions containing polymerase activity were pooled. Thefractions were dialyzed against 20 volumes of column buffer. The pooledfractions were applied to a Toso650Q column (Tosohaas). The column waswashed to baseline OD₂₈₀ and elution effected with a linear 10 columnvolume gradient of 25 mM Tris, pH 7.4, 8% glycerol, 0.5 mM EDTA, 10 mMKCl, 5 mM β-mercaptoethanol to the same buffer plus 650 mM KCl. Activefractions were pooled.

EXAMPLE 7 Characterization of Purified Tne DNA Polymerase

1. Determination of the Molecular Weight of Thermotoga neapolitana DNAPolymerase

The molecular weight of 100 kilodaltons was determined byelectrophoresis in a 12.5% SDS gel by the method of Laemmli, U.K.,Nature (Lond.) 227:680-685 (1970). Proteins were detected by stainingwith Coomassie brilliant blue. A 10 Kd protein ladder (LifeTechnologies, Inc.) was used as standard.

2. Method for Measuring Incorporation of α³⁵ S!-dATP Relative to ³H-dATP

Incorporation of αS!dATP was evaluated in a final volume of 500 μl ofreaction mix, which was preincubated at 72° C. for five minutes,containing either a ³ H!TTP nucleotide cocktail (100 μM each TTP, dATP,dCTP, dGTP with ³ H!TTP at 90.3 cpm/pmol), a nucleotide cocktailcontaining αS!dATP as the only source of dATP (100 μM each αS!dATP,dCTP, dGTP, TTP with α³⁵ S!dATP at 235 cpm/pmol), or a mixed cocktail(50 μM αS!dATP, 50 μM dATP, 100 μM TTP, 100 μM dCTP, 100 μM dGTP with ³⁵αS!dATP at 118 cpm/pmol and ³ H!TTP at 45.2 cpm/pmol). The reaction wasinitiated by the addition of 0.3 units of T. neapolitana DNA polymeraseor T. aquaticus DNA polymerase. At the times indicated a 25 μl aliquotwas removed and quenched by addition of ice cold EDTA to a finalconcentration of 83 mM. 20 μl aliquots of the quenched reaction sampleswere spotted onto GF/C filters. Rates of incorporation were compared andexpressed as a ratio of T. neapolitana to T. aquaticus. Theincorporation of α³⁵ S!dATP by T. neapolitana DNA polymerase wasthree-fold higher than that of T. aquaticus DNA polymerase.

EXAMPLE 8 Reverse Transcriptase Activity

(A)_(n) :(dT)₁₂₋₁₈ is the synthetic template primer used most frequentlyto assay for reverse transcriptase activity of DNA polymerases. It isnot specific for retroviral-like reverse transcriptase, however, beingcopied by many prokaryotic and eukaryotic DNA polymerases (Modak andMarcus, J. Biol. Chem. 252:11-19 (1977); Gerard et al., Biochem.13:1632-1641 (1974); Spadari and Weissbach, J. Biol. Chem. 249:5809-5815(1974)). (A)_(n) :(dT)₁₂₋₁₈ is copied particularly well by cellular,replicative DNA polymerases in the presence of Mn⁺⁺, and much lessefficiently in the presence of Mg⁺⁺ (Modak and Marcus, J. Biol. Chem.252:11-19 (1977); Gerard et al., Biochem. 13:1632-1641 (1974); Spadariand Weissbach, J. Biol. Chem. 249:5809-5815 (1974)). In contrast, mostcellular, replicative DNA polymerases do not copy the synthetic templateprimer (C)_(n) :(dG)₁₂₋₁₈ efficiently in presence of either Mn⁺⁺ orMg⁺⁺, but retroviral reverse transcriptases do. Therefore, in testingfor the reverse transcriptase activity of a DNA polymerase withsynthetic template primers, the stringency of the test increases in thefollowing manner from least to most stringent: (A)_(n) :(dT)₁₂₋₁₈(Mn⁺⁺)<(A)_(n) :(dT)₁₂₋₁₈ (Mg⁺⁺)<<(C)_(n) :(dG)₁₂₋₁₈ (Mn⁺⁺)<(C)_(n):(dG)₁₂₋₁₈ (Mg⁺⁺).

The reverse transcriptase activity of Thermotoga neapolitana (Tne) DNApolymerase was compared with Thermus thermophilus (Tth) DNA polymeraseutilizing both (A)_(n) :(dT)₂₀ and (C)_(n) :(dG)₁₂₋₁₈. Reaction mixtures(50 μl) with (A)_(n) :(dT)₂₀ contained 50 mM Tris-HCl (pH 8.4), 100 μM(A)_(n), 100 μM (dT)₂₀, and either 40 mM KCl, 6 mM MgCl₂, 10 mMdithiothreitol, and 500 μM ³ H!dTTP (85 cpm/pmole), or 100 mM KCl, 1 mMMnCl₂, and 200 μM ³ H!dTTP (92 cpm/pmole). Reaction mixtures (50 μl)with (C)_(n) :(dG)₁₂₋₁₈ contained 50 mM Tris-HCl (pH 8.4), 60 μM(C)_(n), 24 μM (dG)₁₂₋₁₈, and either 50 mM KCl, 10 mM MgCl₂, 10 mMdithiothreitol, and 100 μM ³ H!dGTP (132 cpm/pmole), or 100 mM KCl, 0.5mM MnCl₂, and 200 μM ³ H!dGTP (107 cpm/pmole). Reaction mixtures alsocontained either 2.5 units of the Tth DNA polymerase (Perkin-Elmer) or2.5 units of the Tne DNA polymerase. Incubations were at 45° C. for 10min followed by 75° C. for 20 min.

The table shows the results of determining the relative levels ofincorporation of Tne and Tth DNA polymerase with (A)_(n) :(dT)₂₀ and(C)_(n) :(dG)₁₂₋₁₈ in the presence of Mg⁺⁺ and Mn⁺⁺. Tne DNA polymeraseappears to be a better reverse transcriptase than Tth DNA polymeraseunder reaction conditions more specific for reverse transcriptase, i.e.,in the presence of (A)_(n) :(dT)₂₀ with Mg⁺⁺ and (C)_(n) :(dG)₁₂₋₁₈ withMn⁺⁺ or Mg⁺⁺.

    ______________________________________    DNA Polymerase Activity of Tth and Tne    DNA Polymerase with (A).sub.n :(dT).sub.20 and (C).sub.n :(dG).sub.12-18    DNA Polymerase Activity    (pMoles Complementary  .sup.3 H!dNTP Incorporated)    (A).sub.n :(dT).sub.20  (C).sub.n :(dG)    Enzyme  Mg.sup.++                     Mn.sup.++  Mg.sup.++                                       Mn.sup.++    ______________________________________    Tne     161.8    188.7      0.6    4.2    Tth     44.8     541.8      0      0.9    ______________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 3    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #                23CAGG AAA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1306 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - ATGGCGAGAC TATTTCTCTT TGATGGCACA GCCCTGGCCT ACAGGGCATA TT - #ACGCCCTC      60    - GACAGATCCC TTTCCACATC CACAGGAATT CCAACGAACG CCGTCTATGG CG - #TTGCCAGG     120    - ATGCTCGTTA AATCATAAAG GAACACATTA TACCCCAAAA GGACTACGCG GC - #TGTGGCCT     180    - TCGACAAGAA GGCAGCGACG TTCAGACACA AACTGCTCGT AAGCGACAAG GC - #GCAAAGGC     240    - CAAAGACGCC GGCTCTTCTA GTTCAGCAGC TACCTTACAT CAAGCGGCTG AT - #AGAAGCTC     300    - TTGGTTTCAA AGTGCTGGAG CTGGAAGGGA TACGAAGCAG ACGATATCAT CG - #CCACGCTT     360    - GCAGCAAGGG CTGCACGTTT TTTGATGAGA TTTTCATAAT AACCGGTGAC AA - #GGATATGC     420    - TTCAACTTGT AAACGAGAAG ATAAAGGTCT GGAGAATCGT CAAGGGGATA TC - #GGATCTTG     480    - AGCTTTACGA TTCGAAAAAG GTGAAAGAAA GATACGGTGT GGAACCACAT CA - #GATACCGG     540    - ATCTTCTAGC ACTGACGGGA GACGACATAG ACAACATTCC CGGTGTAACG GG - #AATAGGTG     600    - AAAAGACCGC TGTACAGCTT CTCGGCAAGT ATAGAAATCT TGAATACATT CT - #GGAGCATG     660    - CCCGTGAACT CCCCCAGAGA GTGAGAAAGG CTCTCTTGAG AGACAGGGAA GT - #TGCCATCC     720    - TCAGTAAAAA ACTTGCAACT CTGGTGACGA ACGCACCTGT TGAAGTGGAC TG - #GGAAGAGA     780    - TGAAATACAG AGGATACGAC AAGAGAAAAC TACTTCCGAT ATTGAAAGAA CT - #GGAGTTTG     840    - CTTCCATCAT GAAGGAACTT CAACTGTACG AAGAAGCAGA ACCCACCGGA TA - #CGAAATCG     900    - TGAAGGATCA TAAGACCTTC GAAGATCTCA TCGAAAAGCT GAAGGAGGTT CC - #ATCTTTTG     960    - CCCTGGACCT TGAAACGTCC TCCTTGACCG TTCAACTGTG AGATAGTCGG CA - #TCTCCGTG    1020    - TCGTTTCAAA CCGAAAACAG CTTATTACAT TCCACTTCAT CACAGAACGC CC - #ACAATCTT    1080    - GATGAAACAC TGGTGCTGTC GAAGTTGAAA GAGATCCTCG AAGACCCGTC TT - #CGAAGATT    1140    - GTGGGTCAGA ACCTGAAGTA CGACTACAAG GTTCTTATGG TAAAGGGTAT AT - #CGCCAGTT    1200    - TATCCGCATT TTGACACGAT GATAGCTGCA TATTTGCTGG AGCCAAACGA GA - #AAAATTCA    1260    #               1306TTG AAATTTCTCG GATACAAAAT GACGTC    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 434 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: unknown              (D) TOPOLOGY: unknown    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Met Ala Arg Leu Phe Leu Phe Asp Gly Thr Al - #a Leu Ala Tyr Arg Ala    #                15    - Tyr Tyr Ala Leu Asp Arg Ser Leu Ser Thr Se - #r Thr Gly Ile Pro Thr    #            30    - Asn Ala Val Tyr Gly Val Ala Arg Met Leu Va - #l Ile Ile Lys Glu His    #        45    - Ile Ile Pro Gln Lys Asp Tyr Ala Ala Val Al - #a Phe Asp Lys Lys Ala    #    60    - Ala Thr Phe Arg His Lys Leu Leu Val Ser As - #p Lys Ala Gln Arg Pro    #80    - Lys Thr Pro Ala Leu Leu Val Gln Gln Leu Pr - #o Tyr Ile Lys Arg Leu    #                95    - Ile Glu Ala Leu Gly Phe Lys Val Leu Glu Le - #u Glu Gly Tyr Glu Ala    #           110    - Asp Asp Ile Ile Ala Thr Leu Ala Ser Lys Gl - #y Cys Thr Phe Phe Asp    #       125    - Glu Ile Phe Ile Ile Thr Gly Asp Lys Asp Me - #t Leu Gln Leu Val Asn    #   140    - Glu Lys Ile Lys Val Trp Arg Ile Val Lys Gl - #y Ile Ser Asp Leu Glu    145                 1 - #50                 1 - #55                 1 -    #60    - Leu Tyr Asp Ser Lys Lys Val Lys Glu Arg Ty - #r Gly Val Glu Pro His    #               175    - Gln Ile Pro Asp Leu Leu Ala Leu Thr Gly As - #p Asp Ile Asp Asn Ile    #           190    - Pro Gly Val Thr Gly Ile Gly Glu Lys Thr Al - #a Val Gln Leu Leu Gly    #       205    - Lys Tyr Arg Asn Leu Glu Tyr Ile Leu Glu Hi - #s Ala Arg Glu Leu Pro    #   220    - Gln Arg Val Arg Lys Ala Leu Leu Arg Asp Ar - #g Glu Val Ala Ile Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Ser Lys Lys Leu Ala Thr Leu Val Thr Asn Al - #a Pro Val Glu Val Asp    #               255    - Trp Glu Glu Met Lys Tyr Arg Gly Tyr Asp Ly - #s Arg Lys Leu Leu Pro    #           270    - Ile Leu Lys Glu Leu Glu Phe Ala Ser Ile Me - #t Lys Glu Leu Gln Leu    #       285    - Tyr Glu Glu Ala Glu Pro Thr Gly Tyr Glu Il - #e Val Lys Asp His Lys    #   300    - Thr Phe Glu Asp Leu Ile Glu Lys Leu Lys Gl - #u Val Pro Ser Phe Ala    305                 3 - #10                 3 - #15                 3 -    #20    - Leu Asp Leu Glu Thr Ser Ser Leu Asp Phe As - #n Cys Glu Ile Val Gly    #               335    - Ile Ser Val Ser Phe Lys Pro Lys Thr Ala Ty - #r Tyr Ile Pro Leu His    #           350    - His Arg Asn Ala His Asn Leu Asp Glu Thr Le - #u Val Leu Ser Lys Leu    #       365    - Lys Glu Ile Leu Glu Asp Pro Ser Ser Lys Il - #e Val Gly Gln Asn Leu    #   380    - Lys Tyr Asp Tyr Lys Val Leu Met Val Lys Gl - #y Ile Ser Pro Val Tyr    385                 3 - #90                 3 - #95                 4 -    #00    - Pro His Phe Asp Thr Met Ile Ala Ala Tyr Le - #u Leu Glu Pro Asn Glu    #               415    - Lys Lys Phe Asn Leu Glu Asp Leu Ser Leu Ly - #s Phe Leu Gly Tyr Lys    #           430    - Met Thr    __________________________________________________________________________

What is claimed is:
 1. A substantially pure thermostable Thermotoganeapolitana DNA polymerase, wherein said DNA polymerase is a Pol I-typepolymerase.
 2. The substantially pure thermostable Thermotoganeapolitana DNA polymerase as claimed in claim 1, wherein saidpolymerase has a molecular weight of about 100 kDa.
 3. The substantiallypure thermostable Thermotoga neapolitana DNA polymerase as claimed inclaim 1, wherein said DNA polymerase has the amino acid sequence of theThermotoga neapolitana DNA polymerase produced by Escherichia coliDH10B/pUC-Tne.
 4. A substantially pure Thermotoga neapolitana DNApolymerase having a molecular weight of about 100 kDa, or fragmentsthereof having DNA polymerase activity.
 5. The substantially pureThermotoga neapolitana DNA polymerase or fragments as claimed in claim4, wherein said DNA polymerase has the amino acid sequence of theThermotoga neapolitana DNA polymerase produced by Escherichia coliDH10B/pUC-Tne.
 6. The substantially pure Thermotoga neapolitana DNApolymerase or fragments thereof as claimed in claim 4, wherein said DNApolymerase comprises the amino acid sequence of SEQ ID NO:3.
 7. Thesubstantially pure Thermotoga neapolitana DNA polymerase or fragmentsthereof as claimed in claim 4, wherein said DNA polymerase has reversetranscriptase activity.
 8. The substantially pure Thermotoga neapolitanaDNA polymerase or fragments thereof as claimed in claim 4, wherein saidDNA polymerase has an enhanced rate of incorporation of dATP relative to³⁵ S! Thermus aguaticus DNA polymerase.
 9. A substantially pureThermotoga neapolitana DNA polymerase, wherein said polymerase isencoded by a DNA molecule having the restriction map of the Thermotoganeapolitana DNA polymerase gene as shown in FIG.
 4. 10. A fragment of asubstantially pure Thermotoga neapolitana DNA polymerase, wherein saidpolymerase is encoded by a DNA molecule having the restriction map ofthe Thermotoga neapolitana DNA polymerase gene as shown in FIG. 4 andsaid fragment has DNA polymerase activity.
 11. The fragment of asubstantially pure Thermotoga neapolitana DNA polymerase as claimed inclaim 10, wherein said fragment has reverse transcriptase activity. 12.The fragment of a substantially pure Thermotoga neapolitana DNApolymerase as claimed in claim 10, wherein said fragment has an enhancedrate of incorporation of ³⁵ S! dATP relative to Thermus aquaticus DNApolymerase.
 13. An isolated nucleic acid molecule encoding athermostable Thermotoga neapolitana DNA polymerase, wherein said DNApolymerase is a Pol I-type polymerase.
 14. The isolated nucleic acidmolecule as claimed in claim 13, wherein said nucleic acid moleculeencodes a Thermotoga neapolitana DNA polymerase having a molecularweight of about 100 kDa.
 15. The isolated nucleic acid molecule asclaimed in claim 13, wherein said DNA polymerase has the amino acidsequence of the Thermotoga neapolitana DNA polymerase produced byEscherichia coli DH10B/pUC-Tne.
 16. An isolated nucleic acid moleculeencoding a Thermotoga neapolitana DNA polymerase having a molecularweight of 100 kDa, or fragments thereof having DNA polymerase activity.17. The isolated nucleic acid molecule as claimed in claim 16, whereinsaid DNA polymerase has the amino acid sequence of the Thermotoganeapolitana DNA polymerase produced by Escherichia coli DH10B/pUC-Tne.18. The isolated nucleic acid molecule as claimed in claim 16, whereinsaid DNA polymerase comprises the amino acid sequence of SEQ ID NO:3.19. The isolated nucleic acid molecule as claimed in claim 16, whereinsaid nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO:2.
 20. The isolated nucleic acid molecule as claimed in claim 16,wherein said DNA polymerase has reverse transcriptase activity.
 21. Theisolated nucleic acid molecule as claimed in claim 16, wherein said DNApolymerase has an enhanced rate of incorporation of ³⁵ S! dATP relativeto Thermus aquaticus DNA polymerase.
 22. An isolated nucleic acidmolecule comprising a gene coding for Thermotoga neapolitana DNApolymerase, wherein said gene coding for Thermotoga neapolitana DNApolymerase has the restriction map of the Thermotoga neapolitana DNApolymerase gene as shown in FIG.
 4. 23. An isolated nucleic acidmolecule comprising a fragment of a gene coding for Thermotoganeapolitana DNA polymerase, wherein said gene coding for Thermotoganeapolitana DNA polymerase has the restriction map of the Thermotoganeapolitana DNA polymerase gene as shown in FIG. 4 and said fragmentencodes a polypeptide having DNA polymerase activity.
 24. The isolatednucleic acid molecule as claimed in claim 23, wherein said polypeptidehas reverse transcriptase activity.
 25. The isolated nucleic acidmolecule as claimed in claim 23, wherein said polypeptide has anenhanced rate of incorporation of ³⁵ S!dATP relative to Thermusaguaticus DNA polymerase.
 26. A recombinant nucleic acid molecule,wherein said recombinant nucleic acid molecule comprises thepolynucleotide sequence of an isolated nucleic acid molecule as claimedin any one of claims 13, 16, 22, and
 23. 27. The isolated nucleic acidmolecule as claimed in any one of claims 13, 16, 22, and 23, whereinsaid nucleic acid molecule is a DNA molecule.
 28. The isolated nucleicacid molecule as claimed in claim 27, wherein said DNA molecule furthercomprises a promoter.
 29. The isolated nucleic acid molecule as claimedin claim 28, wherein said promoter is an inducible promoter selectedfrom the group consisting of: a tac promoter, a trp promoter, and a trcpromoter.
 30. A recombinant host cell transformed with a gene encoding athermostable Thermotoga neapolitana DNA polymerase, wherein said DNApolymerase is a Pol I-type polymerase.
 31. A recombinant host celltransformed with a gene encoding a Thermotoga neapolitana DNA polymerasehaving a molecular weight of about 100 kDa, or fragments thereof havingDNA polymerase activity.
 32. The recombinant host cell as claimed inclaim 31, wherein said recombinant host cell can produce a Thermotoganeapolitana DNA polymerase having the amino acid sequence of theThermotoga neapolitana DNA polymerase produced by Escherichia coliDH10B/pUC-Tne.
 33. The recombinant host cell as claimed in claim 31,wherein said recombinant host cell is Escherichia coli DH10B/pUC-Tne.34. The recombinant host cell as claimed in claim 31, wherein saidrecombinant host cell comprises a gene for Thermotoga neapolitana DNApolymerase having the restriction map of the Thermotoga neapolitana DNApolymerase gene as shown in FIG.
 4. 35. The recombinant host cell asclaimed in claim 31, wherein said recombinant host cell comprises afragment of the gene for Thermotoga neapolitana DNA polymerase, whereinsaid DNA polymerase gene has the restriction map of the Thermotoganeapolitana DNA polymerase gene as shown in FIG.
 4. 36. The recombinanthost cell as claimed in any one of claims 30 and 31, wherein said hostcell is prokaryotic.
 37. The recombinant host cell as claimed in claim36, wherein said host cell is Escherichia coli.
 38. The recombinant hostcell as claimed in claim 37, wherein said host cell is Escherichia coliDH10B/pUC-Tne as deposited with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, USDA, underDeposit No. NRRL B-21338.
 39. A method of producing a thermostableThermotoga neapolitana DNA polymerase, said method comprising:(a)culturing a cell transformed with a gene encoding said thermostableThermotoga neapolitana DNA polymerase or fragments thereof having DNApolymerase activity; (b) expressing said gene; and (c) isolating saidDNA polymerase or fragment thereof having DNA polymerase activity fromsaid cell or the cell culture of said cell.
 40. The method of producinga thermostable Thermotoga neapolitana DNA polymerase as claimed in claim39, wherein said cell is a recombinant host cell.
 41. The method ofproducing a thermostable DNA polymerase as claimed in claim 39, whereinsaid thermostable DNA polymerase has a molecular weight of about 100kDa.
 42. The method of producing a thermostable DNA polymerase asclaimed 39, wherein said thermostable DNA polymerase has the amino acidsequence of the Thermotoga neapolitana DNA polymerase produced byEscherichia coli DH10B/pUC-Tne.
 43. The method of producing athermostable DNA polymerase as claimed 40, wherein said recombinant hostcell is Escherichia coli DH10B/pUC-Tne.
 44. The method of producing athermostable DNA polymerase as claimed in claim 39, wherein said genehas the restriction map of the Thermotoga neapolitana DNA polymerase asshown in FIG.
 4. 45. The method of producing a thermostable DNApolymerase as claimed in claim 39, wherein said host cell is aeukaryotic host cell.
 46. The method of producing a thermostable DNApolymerase as claimed in claim 39, wherein said host cell is aprokaryotic host cell.
 47. The method of producing a thermostable DNApolymerase as claimed 46, wherein said prokaryotic host cell isEscherichia coli.